Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) is a ubiquitous enzyme which catalyzes the phosphorylation of adenosine to AMP, using ATP, preferentially, as the phosphate source. Magnesium is also required for the reaction, and the true cosubstrate is probably the MgATP.sup.2- complex (Palella et al., J. Biol. Chem. 1980, 255: 5264-5269). Adenosine kinase has broad tissue and species distribution, and has been isolated from yeast (Leibach et al., Hoppe-Seyler's Z. Physiol. Chem. 1971, 352: 328-344), a variety of mammalian sources (e.g. Miller et al., J. Biol. Chem. 1979, 254: 2339-2345; Palella et al., J. Biol. Chem. 1980, 255: 5264-5269; Yamada et al., Comp. Biochem. Physiol. 1982, 71B: 367-372; Rottlan and Miras-Portugal, Eur. J. Biochem., 1985, 151: 365-371) and certain microorganisms (e.g. Lobelle-Rich and Reeves, Am. J. Trop. Med. Hyg. 1983, 32: 976-979; Datta et al., J. Biol. Chem. 1987, 262: 5515-5521). It has been found to be present in virtually every human tissue assayed including kidney, liver, brain, spleen, placenta and pancreas (Andres and Fox, J. Biol. Chem. 1979, 254: 11388-11393).
Adenosine kinase is a key enzyme in the control of the cellular concentrations of adenosine (Arch and Newsholme, Essays Biochem. 1978, 14: 82-123). Adenosine is a purine nucleoside that is an intermediate in the pathways of purine nucleotide degradation and salvage. In addition, adenosine has many important physiologic effects, many of which are mediated through the activation of specific ectocellular receptors, termed P.sub.1 receptors (Bumstock, in Cell Membrane Receptors for Drugs and Horinones, 1978, (Bolis and Straub, eds) Raven, N.Y. pp. 107-118; Fredholm et al., Pharmacol. Rev. 1994, 46: 143-156). In the central nervous system, adenosine inhibits the release of certain neurotransmiters (Corradetti et al., Eur. J. Pharmacol. 1984, 104: 19-26), stabilizes membrane potential (Rudolphi et al., Cerebrovasc. Brain Metab. Rev. 1992, 4: 346-360), functions as an endogenous anticonvulsant (Dragunow, Trends Pharmacol. Sci. 1986, 7: 128-130) and may have a role as an endogenous neuroprotective agent (Rudolphi et al., Trends Pharmacol. Sci. 1992, 13: 439-445). Adenosine has also been implicated in modulating transmission in pain pathways in the spinal cord (Sawynok et al., Br. J. Pharmacol. 1986, 88: 923-930), and in mediating the analgesic effects of morphine (Sweeney et al., J. Pharmacol. Exp. Ther. 1987, 243: 657-665). In the immune system, adenosine inhibits certain neutrophil functions and exhibits anti-inflammatory effects (Cronstein, J. Appl. Physiol. 1994, 76: 5-13).
Adenosine also exerts a variety of effects on the cardiovascular system, including vasodilation, impairment of atrioventricular conduction and endogenous cardioprotection in myocardial ischemia and reperfusion (Mullane and Williams, in Adenosine and Adenosine Receptors 1990 (Williams, ed) Humana Press, New Jersey, pp. 289-334). The widespread actions of adenosine also include effects on the renal, respiratory, gastrointestinal and reproductive systems, as well as on blood cells and adipocytes.
Endogenous adenosine release appears to have a role as a natural defense mechanism in various pathophysiologic conditions, including cerebral and myocardial ischemia, seizures, pain, inflammation and sepsis. While adenosine is normally present at low levels in the extracellular space, its release is locally enhanced at the site(s) of excessive cellular activity, trauma or metabolic stress. Once in the extracellular space, adenosine activates specific extracellular receptors to elicit a variety of responses which tend to restore cellular function towards normal (Bruns, Nucleosides Nucleotides, 1991, 10: 931-943; Miller and Hsu, J. Neurotrauma, 1992, 9: S563-S577). Adenosine has a half-life measured in seconds in extracellular fluids (Moser et al., Am. J. Physiol. 1989, 25: C799-C806), and its endogenous actions are therefore highly localized.
The inhibition of adenosine kinase can result in augmentation of the local adenosine concentrations at foci of tissue injury, further enhancing cytoprotection. This effect is likely to be most pronounced at tissue sites where trauma results in increased adenosine production, thereby minimizing systemic toxicities. Pharmacologic compounds directed towards adenosine kinase inhibition provide potential effective new therapies for disorders benefited by the site- and event-specific potentiation of adenosine.
Adenosine kinase is also responsible for the activation of many pharmacologically active nucleosides (Miller et al., J. Biol. Chem. 1979, 254: 2339-2345), including tubercidin, formycin, ribavirin, pyrazofurin and 6-(methylmercapto)purine riboside. These purine nucleoside analogs represent an important group of antimetabolites which possess cytotoxic, anticancer and antiviral properties. They serve as substrates for adenosine kinase and are phosphorylated by the enzyme to generate the active form. The loss of adenosine kinase activity has been implicated as a mechanism of cellular resistance to the pharmacologic effects of these nucleoside analogs (e.g. Bennett et al., Mol. Pharmacol., 1966, 2: 432-443; Caldwell et al., Can. J. Biochem., 1967, 45: 735-744; Suttle et al., Europ. J. Cancer, 1981, 17: 43-51). Decreased cellular levels of adenosine kinase have also been associated with resistance to the toxic effects of 2'-deoxyadenosine (Hershfield and Kredich, Proc. Natl. Acad. Sci. USA, 1980, 77: 4292-4296). The accumulation of deoxyadenosine triphosphate (dATP), derived from the phosphorylation of 2'-deoxyadenosine, has been suggested as a toxic mechanism in the immune defect associated with inheritable adenosine deaminase deficiency (Kredich and Hershfield, in The Metabolic Basis of Inherited Diseases, 1989 (Scriver et al., eds), McGraw-Hill, New York, pp. 1045-1075).
Alterations in cellular adenosine kinase activity have also been observed in certain disorders. Adenosine kinase activity was found to be decreased, relative to normal liver, in a variety of rat hepatomas, with activity of the enzyme giving a negative correlation with tumor growth rate (Jackson et al., Br. J. Cancer, 1978, 37: 701-713). Adenosine kinase activity was also diminished in regenerating liver after partial hepatectomy in experimental animals (Jackson et al., Br. J. Cancer, 1978,37: 701-713). Erythrocyte adenosine kinase activity was found to be diminished in patients with gout (Nishizawa et al., Clin. Chim. Acta 1976, 67: 15-20). Lymphocyte adenosine kinase activity was decreased in patients infected with the human immunodeficiency virus (HIV) exhibiting symptoms of AIDS, and increased in asymptomatic HIV-seropositive and HIV-seronegative high-risk subjects, compared to normal healthy controls (Renouf et al., Clin. Chem. 1989, 35: 1478-1481). It has been suggested that measurement of adenosine kinase activity may prove useful in monitoring the clinical progress of patients with HIV infection (Renouf et al., Clin. Chem. 1989, 35: 1478-1481).